The Gram-negative bacteria Yersinia pestis is the causative agent of plague, and is classified as an NIAID category A priority biodefense agent. Y.pestis contains a well-described type III secretion system that has the ability to repress the host responses to the bacteria. However, less is know about immune activation by outer membrane components such as lipopolysaccharide (LPS), how these components interact with the host immune system, and their role in disease progression. Our goal is to define the role of Y. pestis LPS in the development of plague, and furthermore to characterize the impact of interactions by Y.pestis and its LPS with host Toll-like receptors (TLRs) and CD14 during the course of the disease. TLRs and CD14 are central in the innate immune response to microbial challenge. It has recently been suggested that Y.pestis produces a lipid A (main biologically active component of LPS) of lower potency when grown at 37 degrees C (host temperature) compared to 27 degrees C (flea temperature). Our hypothesis is that temperature induced alterations in Y. pestis LPS enable the bacteria to blunt responses mediated by TLR4, contributing to the diminished innate immune responses following infection. We will isolate lipid A from Y. pestis strain KIM (for which the genomic sequence is known) grown at 27xC and 37xC, and characterize the detailed structures. Furthermore, we will analyze the immune activation ability of Y. pestis (grown at 27 degrees C and 37 degrees C) and its LPS with an emphasis on interactions with the TLR signaling pathways, and relate structures to immune activation potential. To investigate the specific role of LPS in disease, we will make bacteria expressing a highly active LPS at 37 degrees C, by over-expressing LPS biosynthesis genes from E.co/i. We will also generate mutants over-expressing the Y.pestis genes, and make bacteria deficient in the same genes. These bacteria will produce altered LPS at both 27 degrees C and 37 degrees C, and we will characterize LPS structure and cell activation potential. To establish the role of Y. pestis LPS in infection in vivo, we will test wild-type and the genetically modified Y. pestis for ability to mount immune activation and infection in wildtype mice and mice genetically deficient for TLRs, MyD88 and CD14. The completion of these studies will provide new information on how Y. pestis interacts with central elements of the innate immune system, knowledge that would be helpful in the development of new therapies for plague and related infections.